Fig. 3: Promoter region hydroxymethylation suppression of AKR1C1 through declined TET1 contributes to brusatol-enhanced progestin sensitivity.

A Transient ectopic expression of NRF2 elevated the levels of TET1 and other target proteins while depletion of NRF2 by siRNAs suppressed its targets. B Keap1 overexpression suppressed NRF2 signaling and TET1. C Schematic diagrams illustrating the construction of TET1-ARE luciferase reporter plasmids with the indicated AREs. D Luciferase activity in endometrial cancer cells transfected with different TET1-ARE constructs after NRF2 overexpression was assessed by a dual luciferase reporter assay. *p < 0.05 compared with the vector control (left). The effect of NRF2 on wild or mutant TET1 ARE’s luciferase activity(right). *p < 0.05, transfected NQO1 ARE served as a positive control. E Brusatol mitigated the effect of NRF2 by decreasing TET1 and AKR1C1 expression. F Ishikawa cells were transfected with TET1 overexpression plasmid. Cells were then treated with or without 20 nM brusatol and the AKR1C1 level was detected by immunoblot. G Ishikawa cells were treated with the indicated dose of brusatol for 48 h, and 5hmC levels in total DNA were detected by a dot blot assay, *p < 0.05 compared with the blank control. H Brusatol abrogated hydroxymethylation in AKR1C1 promoter region. Cells underwent the indicated treatments were subjected to the hMeDIP assay. *p < 0.05. I Keap1 overexpression or TET1 knockdown inhibited the growth of Ishikawa-NRF2 cells, *p < 0.05 compared with the Vector or siCon group.